In spite of a number of advantages of human adenovirus (HAd) vectors as a delivery system for a variety of gene therapy and vaccine applications, the vector immunity is one of the major disadvantages associated with these vectors. This is due to the presence of preexisting HAd-specific immunity in the majority of the human population and the development of a vector-specific immune response following the first inoculation. HAd-specific immunity significantly lowers the efficiency of HAd vector uptake following readministration of the same vector and thus drastically reduces the levels and duration of transgene expression. Another major concern associated with HAd vectors is an acute inflammatory response and hepatotoxicity caused by activation of innate immunity. Our hypothesis is that the primary receptors for HAd5, bovine adenovirus type 3 (BAd3) and porcine adenovirus type 3 (PAd3) are distinct, and therefore, the repertoire of cell types transduced by these adenoviral vectors will be different. This proposal addresses the core issue of eluding vector immunity by sequential administration of human and nonhuman adenoviral vectors. This will avoid toxicity and allow a reduction of the vector dose per inoculation without compromising the therapeutic effect. Under Specific Aim 1, the efficacy of nonhuman adenoviral vectors for gene therapy will be evaluated. In particular, transduction efficiency, the level and persistence of the vector and expression of the vector gene/s will be determined. Specific Aim 2 addresses the role of known adenoviral receptors and co-receptors in internalization of nonhuman adenoviral vectors. Subsequently, a series of in vivo experiments will be conducted to evaluate the usefulness of sequential administration of human and nonhuman adenoviral vectors in circumventing the vector-specific immune response and toxicity (Specific Aim 3). These in vivo experiments will be done in normal immunocompetent mice and major histocompatibility complex (MHC) class I-deficient mice to determine the role of cytotoxic T cells in limiting vector transduction, persistence and biodistribution. Additional experiments will be carried out in a mouse model of breast cancer to determine if differences in gene expression and toxicity occur following intravenous vs. intratumoral inoculation. The successful completion of this project should provide an effective strategy for circumvention of adenoviral vector immunity and toxicity for gene therapy applications.